The process of transcription is essential for all living cells. Transcription is composed of four steps: promoter binding, RNA chain initiation, RNA transcript elongation, and RNA transcript termination. Regulation of transcription can occur at each of these four steps. The long-term research goal of this proposal is to understand the mechanism and the regulation of transcript elongation by eukaryotic RNA polymerases. When eukaryotic genes are activated for transcription, their chromatin structure changes to accommodate transcription factors and to allow efficient transcription by RNA polymerases. However, for many genes there is evidence that transcribed regions are covered with nucleosomes. This raises the questions: (1) How do polymerases transcribe through the chromatin barrier? (2) Can the barrier change the rate of transcript elongation? These questions will be addressed in transcription system in vitro. We will construct homogeneous and well-defined mono-and polynucleosomal chromatin templates. Transcription of these templates will be analyzed using biochemical and molecular genetic techniques, focusing on analysis of eukaryotic RNA polymerases. The specific aims are: (1) To ascertain the mechanism determining the rate of transcription through chromatin by RNA polymerases II and III. Information to be obtained here will provide structural framework for understanding of transcription through chromatin and its rate-limiting step(s). (2) To ascertain the effect of specific elongation factors, modifications of chromatin structure, and modifications of RNA pol II on the rate of transcription through chromatin. These experiments will lead to better understanding of regulation of transcription at the level of transcript elongation. The discovery that some elongation factors play important roles in oncogeneses underscores the potential clinical significance of analysis of the mechanism of transcript elongation.